Histochemical analysis of skeletal muscular tissues of pigs according to genotype MYF 4 ( Short communication )

The results of histochemical analysis of 3 muscles – m. triceps brachii (MTB), m. longissimus thoracitus (MLT) and m. rectus femoris (MRF) – in 2 groups of pigs formed according to the genotypes MYF 4 are presented in this study. Determination of MYF 4 genotypes was performed by the polymerase chain reaction method (PCR). For histochemical analysis, 5 animals detected as homozygotes of the MYF 4-AA type and 5 animals of the heterozygous genotype AB were used out of the total 25 individual animals tested. The histochemical analysis demonstrated that homozygotes AA had larger fat cells on the average than heterozygotes AB in the three studied muscles, which was statistically significant (P<0.05). The percentage surface representation of interstitial tissues was higher in the studied muscles of heterozygotes MYF 4-AB. The volume of interstitial tissues was the highest in the MRF in both groups (myogenin – AA and AB). The average thickness of muscle fibres of the three studied muscles was higher in the homozygote genotype myogenin MYF 4-AA than in heterozygotes MYF 4-AB, which was statistically significant (P<0.05).


Introduction
Striated skeletal muscle is at present a subject of intensive research, as muscles represent one of the most important elements in human nutrition.Studies of muscle growth and meat quality have led to an increasing interest in the microscopic skeletal muscle structure of individual types of animals (KUCHENMEISTER and KUHN 2003, REHFELD et al. 2005, LE FAUCHEUR 2006).The relationship between qualitative and quantitative properties of skeletal muscles and the final meat product is also determined by the growth changes of individual farm animals (CHEREPANOV 2001).Histochemical methods allow us to evaluate the qualitative and quantitative differences in the final product.It is clear that there are definite quantitative differences among the individual farm animals which are under the genetic control of several genes (WIMMERS et al. 2005).One of these is myogenin (MYF 4), which is an essential activator of myogenesis (ERNST et al. 1994, HORAK et al. 2004).We can assume that their effect is correlated with the postnatal growth of skeletal muscles (MAKOVICKÝ 2005).There exists only a little information about the influences of the growth differentiation factor.
The objective of this study was to determine whether there exist some differences between MYF 4 and the postnatal growth of skeletal muscles in pigs.

Material and methods
The Landrace breed of pigs was used for this experiment.They were reared under standard conditions (age, nutrition, stabling, etc).The average weight of the animals at the end of the experiment was 96.3 kg (±3 kg).Immediately after slaughter of the 25 animals we took blood samples in order to be able to detect the genotypes of the MYF 4 gene.We also took samples of three muscle types -m.triceps brachii (MTB), m. longissimus thoracicus (MLT) and m. rectus femoris (MRF).Samples of size 1×1×1 cm were taken at the latest 30 min post mortem.They were then wrapped in aluminium foil Alobal and immediately frozen in liquid nitrogen.One sample was taken from each muscle of the pig.
Genetic analysis of the MYF 4 gene was performed by the PCR technique, and on the basis of the detection of genotypes we performed histochemical analyses of 3 muscle types (MTB, MLT, MRF) from 5 homozygous animals (MYF 4-AA) and 5 heterozygous animals (MYF 4-AB).
Sections 10 μm thick were cut in a freezing microtome (type MTB) at a temperature of -19 °C to -21 °C and subsequently subjected to histological tests.The first series of samples was stained with translucent haematoxylin-eosin, and the second series was stained for succinic dehydrogenase (SDH) activity, in order to detect muscle fibre types.The third series of slices was stained with red oil »0« to detect the neutral lipids.The preparations were evaluated in a light-microscopic picture.The average thickness of muscular fibres, fat cells and the percentage content of interstitial tissues and muscular fibres were measured morphometrically in homozygotes MYF4-AA pigs and heterozygotes MYF 4-AB pigs.10 places in each section were randomly chosen.A minimum of 500 fibres was evaluated from the samples.Objective evaluations were performed with an Olympus Provis light microscope with the software NIS-Elements.The results were evaluated by statistical models.Distinctions among panels were tested using the SAS program with binate-test at the limit α=0.05.

The analysis of fat tissues
We detected fat cells of various sizes and their localization in all three muscles studied in both genotypes (MYF 4-AA and MYF 4-AB).The real and comparative characteristics were achieved on the basis of morphometric measurement of fat cell size.The highest average size value of fat cells in homozygotes MYF 4-AA was in MLT -41.10 μm.The size of fat cells in MTB -39.70 μm was slighty smaller.The fat cells in MRF were considerably smaller, their diameter being 36.70 μm.The minimum and maximum values show that the variation in the size of fat cells in the studied muscles was very constant (Table 1).The size of fat cells in MLT -38.50 μm was again prevalent in the animals detected as heterozygotes AB.The smallest average thickness was in MTB -34.85, μm and in MRF it was slightly higher -35.70 μm.The diameter of fat cells in the three studied muscles in all these animals (AB) was smaller than in homozygotes (AA), but the variability was higher (Table 2).The results demonstrate that there is a statistically significant difference between the size of fat cell of homozygotes MYF 4-AA and the size of fat cells of heterozygotes MYF 4-AB (P<0.05).There is a statistically significant difference between average thickens MTB of homozygotes and MTB of heterozygotes (P<0.05).Differences between MLD of homozygotes and MLD of heterozygotes is statistically not significant (P>0.05).Differences between MRF of homozygotes and MRF of heterozygotes is statistically significant (P<0.05).When evaluating the average size of muscle fibre types, it is possible to state that in all the studied muscles, in both the genotypic groups (AA and AB), the α-white fibres were dominant.We detected the highest values of muscle fibre thickness in MRF for both the genotypes (90.58 μm vs. 86.98μm respectively).The values were higher in all the muscles than the total average size of muscle fibres.We detected the lowest values of muscle fibres with β-red fibres again in all the muscles of both genotypic groups.Their average thickness was at its lowest in MTB (71.36 μm,resp. 66.40 μm).The intermediary α-red fibres had mean values, and their size more or less corresponded to the total average size of muscle fibres (Table 3 and 4).

The analysis of interstitial tissue and muscular fibres
When studying the surface proportion of the individual fibres and interstitial tissues in percentages we performed an additional analysis of muscle structure.It was again demonstrated that the surface proportion of α-white fibres is the highest in all muscle groups in both genotypes MYF 4. The results show that the surface proportion of those fibres in contrast to their size had the lowest values in MRF in both genotypic groups AA and AB (40 % and 46.60 %, respectively).The results suggest that β-red and α-red fibres represent proportionally a grater percentage than α-white, even though they are the thicken ones in terms of diameter (Table 5 and 6).The results indicate that the average thickness of muscular fibres of fat cells of homozygotes MYF 4-AA exceeds the average thickness of muscular fibres of heterozygotes MYF 4-AB in all three muscles.On the basis of genetic analysis, it is necessary to consider other factors while introducing strict control in the animal population.A study performed out by FIEDLER et al. (2001) documents that pigs which were homozygous had a more significant ratio of carcinomatous hyperthermy in comparison to heterozygotes.This is also shown in the case of meat quality.ĽAHUČKÝ et al. (2000) stated that individuals with genetic and metabolic pathological defects should not be used for final hybrid production.OSTROWSKI and BLICHARSKI (1999) investigated histochemical samples of the skeletal muscles from 237 pigs (18 nn, 170 Nn and 49 NN) and did not find any statistically significant difference between the percentage of muscle content, but they demonstrated significant differences of pH in leg muscles after slaughter.NISSEN et al. (2004) divided pigs after weaning into the heaviest, middle-weight and light-weight according to variation of postnatal muscular fibre growth.The differences in muscular fibre growth between light-weight and middle-weight pigs were explained by the larger proportion of muscular fibres in middle-weight pigs.The differences between growth rate of middle-weight and heavyweight pigs were explained by the higher numbers of the same large muscular fibres in the heaviest pigs.The practical value of molecular genetics is underestimated in its underutilization in the economics of rearing (WIMMERS et al. 2005).This fact is a result of poor knowledge of the value and utilization of selection on the basis of markers and their influence on rearing economics.

Table 1
The size of fat cells in μm -homozygotes MYF 4-AA Fettzellgröße in μm bei homozygoten MYF 4-AA Histological analysis showed that the average thickness of muscle fibres varies among the genetically investigated groups.But the thickness tendency in muscles is the same.The homozygotes MYF 4-AA showed higher average values of thickness of muscle fibres in all three muscle types (MTB, MLT, MRF).Those of homozygotes AA had the highest average of muscular fibre thickness in MRF -88.60 μm and the lowest in MTB -73.30 μm.In MLT, muscle fibres had an average thickness of 79.03 μm.The heterozygotes MYF 4-AB had the same tendency of thickness in the direction of MRF (84.72 μm), MLT (75.77 μm) and MTB (69.40 μm) (Table3 and 4).There is a statistically significant difference between average thickness of muscle fibre types of homozygotes MYF 4-AA and the average thickness of muscle fibre types heterozygotes MYF 4-AB (P<0.05).